Several on-going projects will be further developed under this grant. Histidine decarboxylases from several microbial sources will be purified to determine whether they contain pyruvate (like the enzyme from Lactobacillus 30a) or pyridoxal 5'-phosphate at the active center. The two dissimilar chains of the pyruvate enzyme will be separately sequenced; a major effort to obtain crystals of this enzyme suitable for collaborative structural studies of the enzyme will be made, as will efforts to identify, by use of covalent and affinity labelling techniques, the nature of amino acid residues at the active site. Similar studies are planned using the pyridoxal-P enzymes, tryptophanase, histodinol-P aminotransferase and perhaps D-serine dehydratase. All of this work is directed toward a more intimate understanding of the mechanism of action of these enzymes. Ornithine cecarboxylase from Lactobacillus 30a will be purified and characterized to determine whether other enzymes that are pyridoxal-P dependent in most organisms depend upon pyruvate in this organism. An NADP ion-dependent pyridoxine dehydrogenase is being studied in the hope of clarifying its role (none is now known for it) in vitamin B6 metabolism. This enzyme differs from the FAD-dependent 4-pyridoxine dehydrogenase involved in the degradation of vitamin B6. Its purification and characterization with respect to subunit structure, kinetic parameters, substrate specificity, cellular concentration under various conditions of cell growth, and the consequences of its loss through mutation should permit conclusions concerning its role in metabolism to be drawn. Finally, we will continue work on transport mechanisms for vitamin B6, initially attempting to detect and then isolate the periplasmic or membrane-bound receptors for the vitamin. All such transport studies at the cellular level have been conducted with bacteria or yeasts; we can now extend these by similar methods to mammalian cell cultures. As time permits, study of the biosynthesis of vitamin B6 will be reinstituted.